Immunoelectron microscopic analysis of basement membrane components in type VII collagen knockout skin

大疱性营养不良性表皮松解症(RDEB)是一种先天性大疱性疾病，原因是缺乏将表皮基底膜(致密层)固定在真皮上的III型胶原蛋白。III型胶原与其他表皮基底膜分子之间的分子关系尚不完全清楚。本研究的目的是利用RDEB患者的皮肤为此，我们比较了VII型胶原基因敲除的人皮肤和正常人皮肤基底膜分子包括VII型胶原、BPAG2、LN-5的精确超微结构定位。RDEB的诊断经电子显微镜证实，并且缺乏LH7.2抗原和III型胶原的N末端结构域的表达。对5名RDEB患者的血液样本进行基因组DNA洗脱。扩增出编码VII型胶原的COL7A1基因，并对其DNA序列进行分析。在10个等位基因中，发现了4个等位基因的3个突变：6573+1G&GT；C、5504delA和5818delC。皮肤样本取自三名RDEB患者的同意。用包埋后免疫金电子显微镜观察BPAG2的N端、C端和层粘连蛋白5的定位，并与正常人皮肤进行统计学比较。结果表明，BPAG2的N端定位于半桥粒，层粘连蛋白5定位于致密层。这些分子在正常皮肤和RDEB皮肤中的定位没有显著差异。而BPAG2的C末端明显从致密层向表皮侧移动。这些结果表明，VII型胶原可能通过BPAG2的C末端结构域与BPAG2之间存在一定的分子相互作用，这两种作用都定位于正常皮肤的致密层。

Receive dystrophic epidermolysis bullosa (RDEB) is a congenital bullous disease caused by lack of type VII collagen that anchors epidermal basement membrane (lamina densa) to the dermis. Molecular relationship between the type VII collagen and the other epidermal basement membrane molecules is not fully elucidated. The purpose of this study is to elucidate the in vivo molecular ultrastructural relationship between the type VII collagen and the other basement membrane molecules using the skin of RDEB patients, which were the type VII knockout human skin. For this, the precise ultrastructural localization of basement membrane molecules including type VII collagen, BPAG2, laminin 5 were compared between the type VII collagen knockout human skin and the normal human skin. The diagnosis of RDEB confirmed by electron microscopy and the lack of expression of LH7.2 antigen, N-terminus domain of type VII collagen. Blood samples from the five patients with RDEB were processed for genomic DNA elution. COL7A1 gene that encodes type VII collagen was amplified and DNA sequences were analyzed. Among ten alleles, three mutations on four alleles were identified: 6573+1G>C, 5504delA and 5818delC. Skin samples were obtained from three RDEB patients by consent. Localizations of N and C terminus of BPAG2, and laminin 5 were observed with post-embedding immunogold electron microscopy and compared statistically with those of normal human skin. As results, the N terminus of BPAG2 was localized at the hemidesmosome and laminin 5 was localized at lamina densa. There was no significant difference in localization of these molecules between normal and RDEB skin. However, C-terminus of BPAG2 were significantly shifted from lamina densa to the epidermal side. These results indicated that type VII collagen may have some molecular interaction between BPAG2 via C-terminal domain of BPAG2, both of which were localized at lamina densa in the normal skin.